Nitric oxide (NO) has been extensively studied in the setting of ischemia-reperfusion (I/R) injury. Previous studies clearly demonstrate that the deficiency of eNOS exacerbates myocardial I/R injury whereas the overexpression of eNOS, NO donor or inhaled NO gas therapy significantly protect the myocardium. NO possesses a number of physiological properties that makes it a potent cardioprotective-signaling molecule. These include vasodilation and the inhibition of oxidative stress, platelet aggregation, leukocyte chemotaxis and apoptosis. The synthesis of NO is critically influenced by various cofactors such as tetrahydrobiopterin, flavin mononucleotide and flavin adenine dinucleotide, the presence of reduced thiols, and the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA), as well as, substrate and oxygen availability. Without an adequate delivery of substrate and co-factors (conditions that certainly exist in the heart during ischemia), NOS is no longer able to produce NO. Therefore, alternate means to produce NO in ischemic tissues are needed to limit I/R injury. Previous studies in our lab have shown that an acute administration of nitrite protects against myocardial I/R injury. Additionally, our preliminary data demonstrates that modest changes in dietary nitrite intake significantly alter steady-state concentrations of nitrite, nitroso modified proteins, and nitrosyl-heme products and that these biochemical changes have a profound outcome on the severity of acute myocardial infarction. Furthermore, our preliminary data suggests that the observed cardioprotection is mediated, in part, by the reduction of nitrite to NO during both the oral supplementation and ischemic periods. The objective of this proposal is to test the overall hypothesis that nitrite serves as an endogenous storage form of NO that renders cardioprotection through its ability to be converted to NO. To test this hypothesis, we have proposed the following four Specific Aims. Specific Aim 1 will investigate the optimal therapeutic strategy needed to achieve cardioprotection in the setting of myocardial I/R by evaluating different doses and durations of nitrite supplementation. Specific Aim 2 will begin our mechanistic studies of nitrite supplementation therapy by investigating the reduction of nitrite to NO by myoglobin. Specific Aim 3 will investigate some of the molecular mechanisms of nitrite supplementation therapy, including the effects of nitrite on the apoptotic pathway following myocardial I/R. Specific aim 4 will investigate the effects of oral nitrite therapy on the structure and function of mitochondria following myocardial I/R.